P53 Mutant Cell Lines Research Articles

Methylation-regulated ZNF545 inhibits growth of the p53-mutant KYSE150 cell line by inducing p21 and Bax.

The Krüppel-associated box zinc-finger protein 545 (ZNF545) was recently identified as a tumor suppressor in esophageal squamous cell carcinoma (ESCC). However, the role of ZNF545 in the tumorigenesis of esophageal cancer cells expressing loss-of-function mutant p53 has not been elucidated. In the present study, the role of ZNF545 in esophageal tumors and the p53-mutant ESCC cell line, KYSE150, was investigated. ZNF545 mRNA was significantly downregulated in tumors when compared with adjacent normal tissues. Methylated ZNF545 was detected in 76.6% of tumor tissues compared with 28.1% of adjacent normal tissues. Combined pharmacological treatment of KYSE150 cells with a demethylating reagent and deacetylase inhibitor restored the expression of ZNF545. Ectopic expression of ZNF545 activated p53 transcription and upregulated the protein expression levels of pivotal effectors p21 and Bax, which are associated with cell proliferation and apoptosis, respectively, in p53-mutant KYSE150 cells; while suppressing colony formation and inducing apoptosis. ZNF545 was therefore proposed as a potential tumor suppressor responsible for inhibiting the growth of p53-mutant ESCC cell lines. In addition, tumor-specific methylation of ZNF545 may represent an epigenetic diagnostic biomarker and a therapeutic target in patients with esophageal cancer.

Abstract 4441: Discovery of potent and selective inhibitors of USP7 with anti-tumor activity in vitro and in vivo

Abstract USP7 is a deubiquitinase that regulates the levels of multiple downstream targets with roles in cancer progression and immune response. Inhibitors of USP7 may thus decrease oncogene function, increase tumor suppressor function, enhance immune function and sensitize tumor cells to DNA damaging agents. We have discovered a novel chemical series that potently and selectively inhibits USP7 in biochemical and cellular assays. Our inhibitors reduce the viability of multiple p53-wild type cell lines, including several blood cancer and MYCN-amplified neuroblastoma cell lines, as well as a subset of p53-mutant tumor cell lines in vitro. Further, oral administration of our USP7 inhibitors inhibits MM.1S (multiple myeloma; p53-wild type) and H526 (small cell lung cancer; p53-mutant) tumor growth in vivo. Our work confirms that USP7 is a pharmacologically tractable target and future studies will aim to further understand the mechanism of action of USP7 inhibitors in p53-mutant cancers. Citation Format: Yamini M. Ohol, Michael Sun, Paul Leger, Dennis Hu, Berenger Biannic, Payal Rana, Cynthia Cho, Scott Jacobson, Steve Wong, Jerick Sanchez, Xinping Han, Kyle Young, Akinori Okano, Jack Maung, Gene Cutler, Nick Shah, Lavanya Adusumilli, Deepika Kaveri, Oezcan Talay, Deepa Pookot, Betty Abraham, Delia Bradford, Nathan Kozon, Christophe Colas, Andrea Kim, Jacob Schwarz, David Wustrow, Dirk Brockstedt, Paul Kassner. Discovery of potent and selective inhibitors of USP7 with anti-tumor activity in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4441.

P53-Suppressed Oncogene TET1 Prevents Cellular Aging in Lung Cancer.

The role of transcriptional regulator ten-eleven translocation methylcytosine dioxygenease 1 (TET1) has not been well characterized in lung cancer. Here we show that TET1 is overexpressed in adenocarcinoma and squamous cell carcinomas. TET1 knockdown reduced cell growth in vitro and in vivo and induced transcriptome reprogramming independent of its demethylating activity to affect key cancer signaling pathways. Wild-type p53 bound the TET1 promoter to suppress transcription, while p53 transversion mutations were most strongly associated with high TET1 expression. Knockdown of TET1 in p53-mutant cell lines induced senescence through a program involving generalized genomic instability manifested by DNA single- and double-strand breaks and induction of p21 that was synergistic with cisplatin and doxorubicin. These data identify TET1 as an oncogene in lung cancer whose gain of function via loss of p53 may be exploited through targeted therapy-induced senescence. SIGNIFICANCE: These studies identify TET1 as an oncogene in lung cancer whose gain of function following loss of p53 may be exploited by targeted therapy-induced senescence.See related commentary by Kondo, p. 1751.

Invitro study of the Polo-like kinase 1 inhibitor volasertib in non-small-cell lung cancer reveals a role for the tumor suppressor p53.

Polo‐like kinase 1 (Plk1), a master regulator of mitosis and the DNA damage response, is considered to be an intriguing target in the research field of mitotic intervention. The observation that Plk1 is overexpressed in multiple human malignancies, including non‐small‐cell lung cancer (NSCLC), gave rise to the development of several small‐molecule inhibitors. Volasertib, presently the most extensively studied Plk1 inhibitor, has been validated to efficiently reduce tumor growth in preclinical settings. Unfortunately, only modest antitumor activity against solid tumors was reported in clinical trials. This discrepancy prompted research into the identification of predictive biomarkers. In this study, we investigated the therapeutic effect of volasertib monotherapy (i.e., cytotoxicity, cell cycle distribution, apoptotic cell death, cellular senescence, and migration) in a panel of NSCLC cell lines differing in p53 status under both normal and reduced oxygen tension (<0.1% O2). A strong growth inhibitory effect was observed in p53 wild‐type cells (A549 and A549‐NTC), with IC 50 values significantly lower than those in p53 knockdown/mutant cells (A549‐920 and NCI‐H1975) (P < 0.001). While mitotic arrest was significantly greater in cells with nonfunctional p53 (P < 0.005), apoptotic cell death (P < 0.026) and cellular senescence (P < 0.021) were predominantly induced in p53 wild‐type cells. Overall, the therapeutic effect of volasertib was reduced under hypoxia (P < 0.050). Remarkably, volasertib inhibited cell migration in all cell lines tested (P < 0.040), with the exception of for the NCI‐H1975 p53 mutant cell line. In conclusion, our results show an important difference in the therapeutic effect of Plk1 inhibition in NSCLC cells with versus without functional p53. Overall, the p53 wild‐type cell lines were more sensitive to volasertib treatment, suggesting that p53 might be a predictive biomarker for Plk1 inhibition in NSCLC. Moreover, our results pave the way for new combination strategies with Plk1 inhibitors to enhance antitumor activity.

In Vitro Characterization of a Potent p53-MDM2 Inhibitor, RG7112 in Neuroblastoma Cancer Cell Lines.

Background: Neuroblastoma (NB) is one of the most aggressive and common solid tumors in pediatrics. Development of effective new therapeutics for NB is in progress to help reduce mortality and morbidity of the disease, particularly in relapsed patients. The tumor suppressor protein p53 plays a critical role in multiple signaling pathways to maintain cellular hemostasis. Dysregulation of p53 protein and/or molecular aberrations have been associated with multiple human malignancies. p53 stability and protein activity is negatively regulated by the E3 ubiquitin ligase (MDM2). Thus, targeting p53-MDM2 protein-protein interaction is a feasible and promising therapeutic strategy to restore the physiological function of p53 in cancer cells. RG7112 is a highly potent and selective small molecule inhibitor, which target a unique structure located within p53 binding motif of MDM2. Methods: The efficacy of RG7112 in vitro using NB cell lines was examined. Two wild-type (WT)-p53 NB cell lines IMR5 and LAN-5, a mutant p53 cell line SK-N-BE(2), and a WT-p53/p14 deleted cell line SH-EP were employed. Results: Data showed that RG7112 significantly reduced cellular viability of IMR5 (IC50, 562 nM) and LAN-5 (IC50, 430 nM), but not SK-N-BE(2) and SH-EP cells. Further, RG7112 restores p53 and p21 protein levels in IMR5 and LAN-5 in a dose-dependent manner. RG7112 induces cell cycle arresting (60% G1 arresting) in WT-p53 cells (IMR5), but no pronounced effect observed in SK-N-BE(2). In this study, 15 different drugs in combination with RG7112 in IMR5 cell line and identified venetoclax (Bcl-2/Bcl-xL inhibitor) as a promising candidate were evaluated. Conclusions: Taken together, these findings provide initial proof-of-concept data for further investigations of RG7112 in selected subgroups of NB patients.

A HYPOTHESIS OF RADIORESISTANCE AND CELL-SURVIVAL CURVE SHAPE BASED ON CELL-CYCLE PROGRESSION AND DAMAGE TOLERANCE.

Exponential survival curves of early-passage human fibroblasts challenge classic biophysical models of cell inactivation. Thus, X-ray doses of 2-4 Gy inactivate normal, human skin fibroblasts in spite of negligible residual double-strand breaks. By contrast, radioresistant p53-mutant U251 glioblastoma cells proliferate in spite of residual damage. Similarly, p53 wildtype TK6 lymphoblastoid cells show exponential survival curves while the related p53-mutant WTK1 cell line continued to proliferate and showed a shouldered survival curve. Here, we propose a model in which the radioresistant shoulder region is due to tolerance to certain types or amounts of residual damage that would otherwise inactivate normal cells. Thus, the steeper initial slope and absence of a shoulder in the survival curve of normal cells may not imply a higher number of residual lesions but rather non-tolerance to these lesions.

Radiosensitization by irinotecan is attributed to G2/M phase arrest, followed by enhanced apoptosis, probably through the ATM/Chk/Cdc25C/Cdc2 pathway in p53-mutant colorectal cancer cells.

Irinotecan, an analog of camptothecin, which is an inhibitor of topoisomeraseI, is currently used in the treatment of metastatic colorectal cancer. Camptothecin derivatives have been demonstrated to exert radiosensitizing effects on several types of cancer cells. However, to date, at least to the best of our knowledge, few studies have examined these effects in colorectal cancer cell lines. In the present study, we examined the radiosensitizing effects of irinotecan on the p53-mutant colorectal cancer cell lines, HT29 and SW620, and explored the potential underlying mechanisms. Drug cytotoxicity tests revealed that the 24h half-maximal inhibitory concentrations (IC50s) of irinotecan as a single agent were 39.84µg/ml (HT29 95% CI, 38.27-41.48) and 96.86µg/ml (SW620 95% CI, 89.04-105.4); finally, concentrations <2µg/ml were used in the subsequent experiments. Clonogenic assays revealed that irinotecan exerted radiosensitizing effects on the HT29 and SW620 cells, and the sensitivity enhancement ratios (SERs) at 2Gy increased with increasing concentrations (SER at 2Gy, 1.41 for the HT29 cells, 1.87 for the SW620 cells; with irinotecan at 2µg/ml). Subsequently, the cells were divided into 4groups: The control group, irinotecan group, radiation group and combination group. Compared with the control, irinotecan and radiation groups, the combination group had the slowest cell growth rate and the most obvious foci of Ser139p‑γH2AX. Combined treatment resulted in a firstly decreased and then increased M phase arrest and led to the most significant G2/M phase arrest, followed by the most significant increase in apoptosis. The results of western blot analysis indicated that the expression levels of proteins related to the DNA damage response system (Ser1981p‑ATM, Ser345p‑Chk1, Thr68p‑Chk2 and Ser139p‑γH2AX) and the cell cycle (Tyr15p‑Cdc2 and cyclinB1) exhibited the greatest increase in the combined group. In addition, the expression of Ser216p‑Cdc25C was also increased in the combined group, indicating that irinotecan likely radiosensitized the p53-mutant HT29 and SW620 cells through the ATM/Chk/Cdc25C/Cdc2 pathway.

Abstract A06: Cisplatin-induced DNA damage modifies the chromatin landscape of histone H2B monoubiquitination in a p53-dependent manner

Abstract Post-translational modifications (PTMs) of histone tails can function as epigenomic regulators of the cellular response to DNA damage. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is a central histone PTM involved in the DNA damage response. H2Bub1 localizes to double-strand breaks where it functions to decondense chromatin, making it accessible to DNA repair factors. H2Bub1 is also known to be enriched at coding sequences of highly expressed genes, demonstrating the fundamental importance of H2Bub1 for an open chromatin structure that facilitates transcription. H2Bub1 is a dynamic process, written predominantly by the E3 ubiquitin ligase complex of the ring finger proteins RNF20 and RNF40 and erased by multiple deubiquitinases, including those from the ubiquitin-specific peptidase subfamily USP7 and USP44. RNF20 is reported to interact with p53. Platinum drugs are used as standard-of-care for the treatment of high-grade serous ovarian cancer (HGSOC) based on their capacity to damage DNA. We sought to determine the response of ubiquitinated histone tails to a platinum drug with the goal of improving the efficacy of current best practice therapy for HGSOC. Following treatment of a wild-type p53 cell line with an IC75 dose of cisplatin, we combined H2Bub1 chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) to determine genomic enrichment of H2Bub1 in response to DNA damage. Further, we coupled this with RNA-seq (RNA sequencing) to enable correlation of genomic enrichment of H2Bub1 with levels of gene expression. KEGG pathway and gene ontology analyses demonstrated greatest H2Bub1 enrichment in response to DNA damage in p53 signalling pathway genes, including CDKN1A, BBC3, MDM2 and BAX. This correlated with increased expression of p53 target genes in response to DNA damage. Wild-type TP53 itself did not show H2Bub1 enrichment upon treatment with cisplatin. H2Bub1 ChIP experiments were repeated in HGSOC mutant p53 cell line models, specifically OVCAR-3 with the known gain-of-function TP53 mutation R248Q and Kuramochi with the missense TP53 mutation D281Y. H2Bub1 was not enriched in response to DNA damage at wild-type p53 gene targets in these cell lines, nor were these genes highly expressed. We therefore propose that DNA damage induced H2Bub1-enrichment in mutant p53 cell lines will have an entirely distinct genomic profile compared to wild-type p53 cell lines, with important roles in enabling the expression of mutant p53 target genes in HGSOC. Experiments are currently under way to directly address this hypothesis. Lastly, we have used clonogenic cell survival assays to demonstrate that downregulation of RNF20, either transiently using short interfering (si) RNA or stably,using short hairpin (sh) RNA, decreased the ability of HGSOC cells to survive treatment with cisplatin. Down-regulation of H2Bub1 by downregulation of its E3 ubiquitin ligase RNF20 may represent a novel treatment strategy to increase the efficacy of DNA damage-based therapy for HGSOC. Citation Format: Alexander J. Cole, Kristie-Ann Dickson, Christopher Liddle, Clare Stirzaker, Roderick Clifton-Bligh, Deborah J. Marsh. Cisplatin-induced DNA damage modifies the chromatin landscape of histone H2B monoubiquitination in a p53-dependent manner. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A06.

Abstract B27: Transfection of mutant P53 promotes membrane translocation of GLUT1 and increases glucose uptake in MCF10A human breast epithelial cells

Abstract Background: High rate of glycolysis exhibited by cancer cells plays a crucial role in carcinogenesis and tumor progression. Previous study showed that gain-of-function (GOF) by mutant P53 drives the Warburg effect via GLUT1 membrane translocation in malignancies. Also, in the patients with a preoperative 18F-fluorodeoxy glucose positron emission tomography (FDG-PET), which enables to estimate glucose uptake by tumor as standardized uptake value (SUV), we identified high SUV as a poor prognostic marker. In this study, we wondered whether P53 promotes GLUT1 membrane translocation and increases glucose uptake in human normal breast epithelial cells using MCF10A cells. Methods: We generated mutant-P53 MCF10A cell line, which include sequence variants (R175H, R273H, and R248W). First, to generate stable P53 knockdown cell lines, lentiviral transduction particles from TRC-pLKO-U6 shP53 were infected into MCF10A. To generate mutant P53 producing MCF10A, pcDNA mutant P53 was transfected using lipofectamin (Invitrogen) into stable P53 knockdown cell lines. Glucose uptake assay and confocal imaging studies with 2NDBG were performed. Confocal imaging system was used to detect GLUT1 membrane expression. In case-matched patients according to P53 mutation (n=114), we compared SUV. Mutational analysis of exons 5-9 of the P53 gene was carried out using Sanger sequencing. Results: Glucose uptake assay showed that the level of glucose uptake was increased in P53 mutant MCF10A cell lines (R175H or R273H, and R248W) with a 1.5-1.8 fold change compared with wild-type MCF10A. The increase of glucose uptake by mutant-P53 was reproducible with the experiments with 2NBGD. The mRNA expressional level of LDHA was also upregulated by mutant P53. In contrast, the mRNA expression of GLUT1, hexokinase 2, PKM1, PKM2, and G6PD was not elevated by P53 mutation. The confocal imaging studies captured translocation to membrane of GLUT1 in mutant P53-MCF10A. However, in wild-type MCF10A, GLUT1 was diffusely expressed in cytoplasm. In patients, the mean SUV of p53-mutational group was significantly higher than that of wild-type p53-group (7.49 vs. 5.44, P=0.013). Compared with the group with intact p53, recurrence-free survival (RFS) was significantly reduced in the group with p53 mutation (P =0.017). In multivariate analysis, p53 mutational status carried prognostic significance (hazard ratio 3.73, 95% CI 1.15-12.07) independent of tumor size, nodal status, and estrogen receptor status. Conclusion: We showed that P53 mutation promotes GLUT1 translocation to membrane, which consequently induces glucose influx in MCF10A cells, elucidating that P53 mutation contributes to the increase of SUV in patients with breast cancer. Citation Format: Sung Gwe . Ahn, Hyekyoung Shin, Bae Soong June, Yoon Changik, Lee Hak Woo, Jeong Joon. Transfection of mutant P53 promotes membrane translocation of GLUT1 and increases glucose uptake in MCF10A human breast epithelial cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B27.

Abstract 3356: The EWS/FLI1 transcriptome is characterized by marked heterogeneity across Ewing sarcoma cell lines

Abstract BACKGROUND: Ewing sarcoma (ES) is the second most common pediatric bone cancer. ES cells are dependent on the activity of the EWS/FLI1 transcription factor for cell survival. EWS/FLI1 drives proliferation, dysregulates the cell cycle and establishes a block in differentiation. Despite the known dependence of Ewing sarcoma on EWS/FLI1, the exact identity of all the downstream targets of EWS/FLI1 is not known. While gene signatures have been reported for EWS/FLI1, these fail to capture the marked heterogeneity in the EWS/FLI1 transcriptome or correlate these expression changes with cellular phenotype. METHODS: Next generation RNA sequencing with deep coverage was used to generate a comprehensive analysis of the EWS/FLI1 transcriptome by silencing EWS/FLI1 in 5 different ES cell lines at two different time points. These results were organized into gene expression networks and signaling nodes were identified by weighted correlation network analysis (WGCNA). The networks were validated by a 500-gene signature of EWS/FLI1 targets using a next generation sequencing capture assay and the Illumina platform. The cellular endpoint of EWS/FLI1 silencing was evaluated by caspase 3,7 cleavage to measure apoptosis, BGAL staining to measure senescence and scratch assays to capture migration. RESULTS: Next generation RNA sequencing with siRNA silencing of EWS/FLI1 revealed marked heterogeneity in the EWS/FLI1 transcriptome among cell lines. Targets of EWS/FLI1 that were common across cell lines exhibited small magnitude gene expression changes (log fold &amp;lt;2), while large magnitude gene expression changes were unique to individual cell lines (log fold&amp;gt;2). When the log fold change in expression was restricted to greater than 2, only 13 common repressed targets and 4 common induced targets were identified in all 5 cell lines. In addition, silencing of EWS/FLI1 induced dramatically different phenotypes across cell lines, where p53 wild-type cell lines underwent senescence and the p53 mutant cell lines did not. Most cell lines did not continue to proliferate with EWS/FLI1 silencing with the exception being the widely used A673 cell line. Cellular migration did not change with EWS/FLI1 silencing in many of the cell lines in contrast to A673 cells. CONCLUSIONS: EWS/FLI1 drives different gene expression signatures and different phenotypes in unique cell lines. This diversity needs to be considered as EWS/FLI1 directed therapies are developed. Citation Format: Susan M. Kitchen-Goosen, Megan J. Bowman, Marie Adams, Penny Berger, Patrick J. Grohar. The EWS/FLI1 transcriptome is characterized by marked heterogeneity across Ewing sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3356.

Cytotoxic Profiling of Endogenous Metabolites Relevant to Chronic Fatigue Immune Dysfunction Syndrome (CFIDS) on p53 Variant Human Colon Carcinoma Cell Lines.

Chemoprophylatic strategies against development of multifactorial diseases utilize compounds to block the multistep events in chronic inflammation and carcinogenesis. The successful chemopreventative candidate must therefore selectively inhibit growth of transformed cells and be administered frequently to confer maximal protection with minimal side effects. In addition to synthetic and exogenous natural compounds, endogenous metabolites represent another class of compounds that exhibit anticarcinogenic and anti-inflammatory properties contributing to proper cell function. To assess the effectiveness of these compounds warrants an understanding of their cytotoxic mode of action. In this study, p53 variant human colon carcinoma cell lines were chronically exposed to varying concentrations of the endogenous metabolites—phenyl acetate, ursodeoxycholate, and tauroursodeoxycholate—to determine the role of p53-induced cytotoxicity, with p53 mutant and deficient cell lines representing precancerous lesions. Cytotoxicity was assessed using clonogenic assays, and macroscopic colony counts were used to quantify cell survival. The results demonstrate that the bile acids, ursodeoxycholate and tauroursodeoxycholate, exhibit selective cytotoxicity toward nonfunctional p53 cell lines suggesting a p53-mediated role in inhibition of cell clonogenicity and potential chemopreventative properties. Although each compound displays this described effect, the tauroursodeoxycholate demonstrates high significance suggesting it might have practical uses in vivo.

Abstract 3982: Dual inhibition of HDAC and PI3 kinase signaling pathways with CUDC-907 in treatment of p53-driven medulloblastoma

Abstract Comprehensive studies of the medulloblastoma (MB) genome, epigenome and transcriptome have placed MB in four molecular subgroups: WNT, SHH, and two non-WNT/non-SHH: Group 3 and Group 4. Furthermore, studies suggest that p53-mutated SHH MBs frequently harbor genetic anomalies involving MYCN and GLI2 amplifications, which confer poor prognosis and resistance to treatments. The phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) intracellular signaling pathway that regulates cell growth, survival and invasion, is commonly deregulated in MB. Therefore, the PI3K/Akt/mTOR pathway may be a suitable target for therapeutic intervention. Moreover, Histone deacetylases (HDAC) regulate cancer initiation and progression, and studies have shown that small molecule inhibitors of HDAC can effectively limit MB proliferation. We aim to determine the status of tumor suppressor gene p53 in defining metastatic potential in SHH-MB and to establish the therapeutic efficacy of targeting these pathways in p53-driven MB cells with a dual agent or individual agents. Immunohistochemistry and FISH were used to determine the status of mutant p53 and MYC amplification or iso-p53, respectively, in MB tumors (n=41). p53-mutant MB cell line was used to investigate the signaling pathway that regulates proliferation, migration, and drug resistance using inhibitors of HDAC (LBH-589) and PI3K/mTOR (BKM-120/rapamycin) or CUDC-907 (dual inhibitor of HDACs and the PI3K/AKT). Results demonstrated that: 1) GAB-1 was highly expressed in the Shh group (82%) and KV1 expression was evenly distributed in all subtypes; 2) loss of p53 and overexpression of MYC varied in each subtype, but did not correlate with metastasis; 3) combined treatment with LBH-589 and BKM-120 or single treatment with CUDC-907 reduced cell proliferation, migration and S-phase entry; 4) MB cells were resistant to BKM -120, while LBH-589 or CUDC-907 caused apoptosis; 5) tumor formation was suppressed by BKM-120 given with mTOR inhibitors; 6) AKT and 4E-BP1 dephosphorylated following treatments with BKM-120 or CUDC-907. In conclusion, despite current clinical limitations, dual HDAC and PI3K inhibitors may benefit the understanding of aberrant signaling pathways in genetically driven MB, and may provide basis for future targeted therapies. Citation Format: Jennifer S. Ronecker, Julia Pazniokas, Anubhav G. Amin, Raphael Salles Scortegagna de Medeiros, Sidnei Epelman, Chirag Ghandi, Nelci Zanon, Meic H. Schmidt, Meena Jhanwar-Uniyal. Dual inhibition of HDAC and PI3 kinase signaling pathways with CUDC-907 in treatment of p53-driven medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3982.

Abstract CT033: Safety and early efficacy signals for COTI-2, an orally available small molecule targeting p53, in a phase I trial of recurrent gynecologic cancer

Abstract Background: Mutated p53 tumor suppressor protein is involved in &amp;gt;50% of all malignancies; however, only recently have candidate drugs designed to target p53 mutations entered clinical trials. COTI-2, an orally bioavailable "third-generation" thiosemicarbazone, was shown to restore the structure and function of mutant p53 proteins and induced growth inhibition in a number of p53 mutant cell lines. Thus, a three-part phase I dose escalation trial of COTI-2 was initiated with Part 1 reported herein. Methods: A multicenter, open label, 3+3 dose escalation trial enrolling patients with recurrent gynecological cancers with adequate performance status and bone marrow function. Patients could have unlimited prior therapies. Patients received COTI-2 5x/week orally over a 28-day cycle. Disease response was assessed by RECIST 1.1 every 8 weeks. Results: Twenty-four patients received study treatment: 19 ovarian, 3 cervical, and 2 endometrial cancers. Median age was 60 years (45-73 yrs) and patients had received a median of 5 prior chemotherapy regimens. After 4 dose cohorts (0.25, 0.5, 1.0, and 1.7 mg/kg), the Recommended phase II Dose (R2PD) for gynecologic cancer was 1.0 mg/kg. Dose-limiting toxicities (DLT) were encountered at 1.0 mg/kg (G3 abdominal pain, G3 sensory peripheral neuropathy, n=1) and 1.7 mg/kg, (G3 neuralgia n=1, G3 myalgia n=1). All DLTs resolved off treatment. Most common adverse events (&amp;gt; 10%) were nausea (67%), vomiting (67%), fatigue (54%), abdominal pain (46%), constipation (29%), anemia (29%), dyspnea (29%), anorexia (29%), urinary tract infection (21%), and hypokalemia (20%), myalgia (16%), diarrhea (16%), pyrexia (17%), peripheral neuropathy (17%), increased creatinine (13%), and weight loss (13%). Pharmacokinetic assessment showed oral administration of COTI-2 yielded a Tmax between 15-90 minutes and a half-life of 8-10 hours. Fifteen patients were evaluable for efficacy, having received at least one cycle of treatment. Of these, 13 had ovarian, 1 had cervical, and 1 had endometrial cancer. Eight patients had received one or more bevacizumab combinations, and two had received immune checkpoint inhibitors. Following COTI-2 treatment, 10 patients showed signs of possible activity: 1 patient had stable disease, 4 had stable target lesions, and 5 had stable non-target lesions. Twelve patients had comprehensive genomic profiling by next generation sequencing: 3 had wild-type p53 and 9 had either hotspot or non-hotspot mutations. Conclusions: COTI-2 was deemed generally safe and well-tolerated. Part 2 of the study is currently underway in HNSCC patients, with additional data expected in 2018, and Part 3 will follow with expansion to combination treatment of ovarian and HNSCC patients. Clinical trial information: NCT02433626. Citation Format: Shannon N. Westin, Wilberto Nieves-Neira, Christian Lynam, Kowthar Y. Salim, Alison D. Silva, Richard T. Ho, Gordon B. Mills, Robert L. Coleman, Filip Janku, Daniela Matei. Safety and early efficacy signals for COTI-2, an orally available small molecule targeting p53, in a phase I trial of recurrent gynecologic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT033.

Abstract 3954: Anti-cancer agent P306 restores p53 pathway through PUMA

Abstract The tumor-suppressor p53 prevents cancer development via initiating cell-cycle arrest, cell death, repair, or anti-angiogenesis processes. Over 50% of human cancers harbor cancer-causing mutations in p53. p53 mutations not only abrogate its tumor-suppressor function, but also endow mutant p53 with a gain of function (GOF), creating a proto-oncogene that contributes to tumorigenesis, tumor progression, and chemo- or radiotherapy resistance. Thus, targeting mutant p53 and/or restoring a wild-type p53 signaling pathway provides an attractive strategy for cancer therapy. We previously showed that P01, a natural product, has potent anti-cancer activity against tumors with mutated p53. Based on the structure of the pharmacophore of the P01 family, we designed and synthesized new analogues. The novel synthetic analogue, P306, is potent in reducing both short-term and long-term proliferation in a broad panel of mutant p53 cell lines, including HT29, SW480, DLD-1, MDA-MB-231, MDA-MB-468, U251, FaDu, CAL-27, PANC-1, ASPC-1 and H1975, with IC50 values in the range of 33.9 to 242 nM. In this study, we have characterized a distinct mechanism of P306 that engages the apoptosis pathway by upregulating pro-apoptotic proteins including PUMA in a time-and dose-dependent manner in colorectal cancer cell lines. PUMA is necessary for the apoptotic effects of P306 in colorectal cancer cells with PUMA-mediated caspase 8 activation mediating P306-induced apoptosis. P306 not only restores p53 pathway but also depletes mutant p53 protein in various mutant p53-expressing cancer cells regardless of what kinds of mutation. P306 treatment potently downregulates c-Myc protein level, and apoptosis induced by P306 is dependent on the c-Myc-PUMA pathway. Our results provide novel mechanistic insights into the tumor cell death promoting activity of a candidate therapeutic agent for cancer. Citation Format: Xiaobing Tian, Amriti R. Lulla, Shengling Zhang, Avital Lev, Philip Abbosh, Rahmat Sikder, David T. Dicker, Wafik S. El-Deiry. Anti-cancer agent P306 restores p53 pathway through PUMA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3954.

Functional Precision Medicine Identifies Novel Druggable Targets and Therapeutic Options in Head and Neck Cancer.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with high mortality and a lack of targeted therapies. To identify and prioritize druggable targets, we performed genome analysis together with genome-scale siRNA and oncology drug profiling using low-passage tumor cells derived from a patient with treatment-resistant HPV-negative HNSCC.Experimental Design: A tumor cell culture was established and subjected to whole-exome sequencing, RNA sequencing, comparative genome hybridization, and high-throughput phenotyping with a siRNA library covering the druggable genome and an oncology drug library. Secondary screens of candidate target genes were performed on the primary tumor cells and two nontumorigenic keratinocyte cell cultures for validation and to assess cancer specificity. siRNA screens of the kinome on two isogenic pairs of p53-mutated HNSCC cell lines were used to determine generalizability. Clinical utility was addressed by performing drug screens on two additional HNSCC cell cultures derived from patients enrolled in a clinical trial.Results: Many of the identified copy number aberrations and somatic mutations in the primary tumor were typical of HPV(-) HNSCC, but none pointed to obvious therapeutic choices. In contrast, siRNA profiling identified 391 candidate target genes, 35 of which were preferentially lethal to cancer cells, most of which were not genomically altered. Chemotherapies and targeted agents with strong tumor-specific activities corroborated the siRNA profiling results and included drugs that targeted the mitotic spindle, the proteasome, and G2-M kinases WEE1 and CHK1 We also show the feasibility of ex vivo drug profiling for patients enrolled in a clinical trial.Conclusions: High-throughput phenotyping with siRNA and drug libraries using patient-derived tumor cells prioritizes mutated driver genes and identifies novel drug targets not revealed by genomic profiling. Functional profiling is a promising adjunct to DNA sequencing for precision oncology. Clin Cancer Res; 24(12); 2828-43. ©2018 AACR.

Endogenous Leu332Gln mutation in p53 disrupts the tetramerization ability in a canine mammary gland tumor cell line.

Mutations in the p53 gene are associated with more than half of all human cancers. These mutations often cause a disruption of the tumor-suppressor function of p53 and induce genomic instabilities. Wild‑type p53 requires tetramerization to function as an initiator of cell cycle arrest and apoptosis. Although alterations in p53 tetramerization caused by mutation have been well studied, there are few cell lines containing an endogenous mutation in the tetramerization domain of p53. Here, we report the discovery of a canine mammary gland tumor cell line CTB‑m2, which contains the Leu332Gln (L332Q) mutation corresponding to Leu344 in the tetramerization domain of human p53. Although CTB‑m2 cells are genetically heterozygous for the Leu332Gln mutation, the mutant mRNA was almost exclusively expressed. CTB‑m2 cells showed enhanced cell proliferation compared to wild‑type p53-expressing CTB‑m cells of the same lineage. A p53 tetramerization reporter assay showed that the ability of the p53 mutant to form tetramers was significantly lower than that of wild‑type p53. An immunoblot analysis of cross-linked p53 oligomerized forms demonstrated that the L332Q mutant lacked the ability to form tetramers but retained the ability to form dimers. These data suggest that the p53 mutant cell line CTB‑m2 could be a useful tool for analyzing the precise tetramerization mechanisms of p53 and verifying the effects of therapeutic agents against tumors expressing p53 mutants that lack the ability to tetramerize.

Cepharanthine exhibits a potent anticancer activity in p53-mutated colorectal cancer cells through upregulation of p21Waf1/Cip1.

Cepharanthine(CEP), a biscoclurine alkaloid isolated from StephaniacepharanthaHayata, has demonstrated anticancer activity in several different types of cancer cells. Colorectal cancer(CRC) is one of the most common cancers in both men and women. Mutated p53 in CRC was reported to be associated with resistance to commonly used chemotherapeutic agents including, 5‑fluorouracil, oxaliplatin and irinotecan. Many studies reported that mutation of p53 induced chemoresistance through several mechanisms, including induction of drug efflux, disruption of cell cycle regulation, evasion of apoptosis and upregulation of DNA repair. This study aimed to evaluate the anticancer activity of CEP in p53 mutant versus p53 wild-type colorectal cancer cells and determine its underlying mechanisms of action. Our results showed that CEP induced colorectal cancer cell death in a concentration-dependent manner. Remarkably, CEP was more effective in controlling the growth of the p53 mutant colorectal cancer cell lines, HT‑29 and SW-620, than the p53 wild-type colorectal cancer cell lines, COLO‑205 and HCT-116. Further studies on the underlying mechanisms revealed that CEP could induce cell cycle arrest and apoptosis in both HT‑29 and COLO‑205 cells. Treatment with CEP dramatically increased p21Waf1/Cip1 expression levels of the p53 mutant cell line HT‑29 and to a lesser extent, the p53 wild-type cell line COLO‑205. In addition, cyclinA and Bcl‑2 expression levels of both cell lines were significantly downregulated following treatment with CEP. CEP also induced ROS formation in colorectal cancer cells. Taken together, we concluded that CEP effectively induced cell cycle arrest and apoptosis which may be mediated through upregulation of p21Waf1/Cip1, downregulation of cyclinA and Bcl‑2 and induction of ROS production in colorectal cancer cells. These findings suggested that CEP could potentially be a novel anticancer agent for p53 mutant colorectal cancer cells which are often resistant to current chemotherapeutic agents.

Mutant p53 as a therapeutic target for the treatment of triple-negative breast cancer: Preclinical investigation with the anti-p53 drug, PK11007

The identification of a targeted therapy for patients with triple-negative breast cancer (TNBC) is one of the most urgent needs in breast cancer therapeutics. The p53 gene is mutated in approximately 80% of patients with TNBC, and is a potential therapeutic target for patients with this form of breast cancer. The 2-sulfonylpyrimidine compound, PK11007, preferentially decreases viability in p53-compromised cancer cell lines. We investigated PK11007 as a potential new treatment for TNBC. IC50 values for inhibition of proliferation in a panel of 17 breast cell lines by PK11007 ranged from 2.3 to 42.2 μM. There were significantly lower IC50 values for TNBC than for non-TNBC cell lines (p = 0.03) and for p53-mutated cell lines compared with p53 WT cells (p = 0.003). Response to PK11007 however, was independent of the estrogen receptor (ER) or HER2 status of the cell lines. In addition to inhibiting cell proliferation, PK11007 induced apoptosis in p53 mutant cell lines. Using RNAseq and gene ontology analysis, we found that PK11007 altered the expression of genes enriched in pathways involved in regulated cell death, regulation of apoptosis, signal transduction, protein refolding and locomotion. The observations that PK11007 inhibited cell proliferation, induced apoptosis and altered genes involved in cell death are all consistent with the ability of PK11007 to reactivate mutant p53. Based on our data, we conclude that targeting mutant p53 with PK11007 is a potential approach for treating p53-mutated breast cancer, including the subgroup with TN disease.

Cytotoxic Profiling of Plant Secondary Metabolites on P53 Variant Human Colon Carcinoma Cell Lines

Chemoprevention strategies employ the use of compounds to inhibit the initiation, promotion, and progression phases of carcinogenesis. The successful chemopreventative candidate must therefore (1) selectively inhibit growth of transformed cells and (2) be administered on a frequent basis to confer maximal protection. Phytochemicals are a subclass of bioactive plant secondary metabolites that exhibit antioxidative, anticarcinogenic, and anti-inflammatory properties contributing to proper cell function. To assess the effectiveness of these compounds warrants an understanding of their cytotoxic mode of action. In this study, p53 variant human colon carcinoma cell lines were chronically exposed to varying concentrations of the phytochemicals—curcumin, andrographolide, and d-limonene—to determine the role of p53-induced cytotoxicity, with p53-mutant and p53-deficient cell lines representing precancerous lesions. Cytotoxicity was assessed using clonogenic assays and macroscopic colony counts were used to quantify cell survival. The results demonstrate that each phytochemical exhibits selective cytotoxicity toward nonfunctional p53 cell lines, suggesting a p53-mediated role in inhibition of cell clonogenicity and potential chemopreventative properties. Although each compound displays this described effect, only the d-limonene demonstrates considerable chemoprotection, suggesting it might have practical implications in vivo.

Mechanisms of Targeting the MDM2-p53-FOXM1 Axis in Well-Differentiated Intestinal Neuroendocrine Tumors

Background/Aims: The tumor suppressor p53 is rarely mutated in gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) but they frequently show a strong expression of negative regulators of p53, rendering these tumors excellent targets for a p53 recovery therapy. Therefore, we analyzed the mechanisms of a p53 recovery therapy on intestinal neuroendocrine tumors in vitro and in vivo.Methods: By Western blot and immunohistochemistry, we found that in GEP-NEN biopsy material overexpression of MDM2 was present in intestinal NEN. Therefore, we analyzed the effect of a small-molecule inhibitor, nutlin-3a, in p53 wild-type and mutant GEP-NEN cell lines by proliferation assay, flow cytometry, immunofluorescence, Western blot, and by multiplex gene expression analysis. Finally, we analyzed the antitumor effect of nutlin-3a in a xenograft mouse model in vivo. During the study, the tumor volume was determined. Results: The midgut wild-type cell line KRJ-I responded to the treatment with cell cycle arrest and apoptosis. By gene expression analysis, we could demonstrate that nutlins reactivated an antiproliferative p53 response. KRJ-I-derived xenograft tumors showed a significantly decreased tumor growth upon treatment with nutlin-3a in vivo. Furthermore, our data suggest that MDM2 also influences the expression of the oncogene FOXM1 in a p53-independent manner. Subsequently, a combined treatment of nutlin-3a and cisplatin (as chemoresistance model) resulted in synergistically enhanced antiproliferative effects. Conclusion: In summary, MDM2 overexpression is a frequent event in p53 wild-type intestinal neuroendocrine neoplasms and therefore recovery of a p53 response might be a novel personalized treatment approach in these tumors.